TGF-β1-Induced Connective Tissue Growth Factor (CCN2) Expression in Human Renal Proximal Tubule Epithelial Cells Requires Ras/MEK/ERK and Smad Signalling

Abstract

Background: Connective tissue growth factor (CTGF, CCN2) plays a fundamental role in the development of tissue fibrosis by stimulating matrix deposition and mediating many of the pro-fibrotic effects of transforming growth factor (TGF)-β. CCN2 induction by TGF-β in renal proximal tubule epithelial cells (PTECs) is likely to play an important role in the development of tubulointerstitial fibrosis. In this study, we investigated the induction of CCN2 by TGF-β1 and the possible mechanisms of this induction in human PTECs. Methods: Experiments were performed on primary and transformed (human kidney cell (HKC)-clone 8) human PTECs. Induction of CCN2 in response to TGF-β1 was studied at the gene promoter level by reporter gene assay, mRNA by semi-quantitative RT-PCR and protein by immunoblotting. While chemical inhibitors were used to assess the role of Ras/MEK/ERK1,2 signalling, an HKC cell line over-expressing Smad7 was used to assess the role of Smad signalling in induction of CCN2 by TGF-β1. Results: TGF-β1 induced CCN2 promoter activity, mRNA and protein in human PTECs. TGF-β1-dependent CCN2 promoter activity was reduced by inhibiting Ras and MEK activation. MEK inhibition also resulted in inhibition of the TGF-β1-induced secreted CCN2 protein. There was no significant increase in CCN2 gene promoter activity or protein by TGF-β1 in Smad7 over-expressing HKCs. Conclusions: TGF-β1 induces the expression of CCN2 in human PTECs. This induction is dependent on Ras/MEK/ERK and Smad signalling. Inhibiting TGF-β induced CCN2 by targeting Smad and/or Ras/MEK/ERK1,2 signalling pathways could be of therapeutic value in renal fibrosis.